Abstract
Plants adapt to heat via thermotolerance pathways in which the activation of protein folding chaperones is essential. In eukaryotes, protein disulfide isomerases (PDIs) facilitate the folding of nascent and misfolded proteins in the secretory pathway by catalyzing the formation and isomerization of disulfide bonds and serving as molecular chaperones. In Arabidopsis, several members of the PDI family are upregulated in response to chemical inducers of the unfolded protein response (UPR), including both members of the non-classical PDI-M subfamily, PDI9 and PDI10. Unlike classical PDIs, which have two catalytic thioredoxin (TRX) domains separated by two non-catalytic TRX-fold domains, PDI-M isoforms are orthologs of mammalian P5/PDIA6 and possess two tandem catalytic domains. Here, PDI9 accumulation was found to be upregulated in pollen in response to heat stress. Histochemical staining of plants harboring the PDI9 and PDI10 promoters fused to the gusA gene indicated they were actively expressed in the anthers of flowers, specifically in the pollen and tapetum. Immunoelectron microscopy revealed that PDI9 localized to the endoplasmic reticulum in root and pollen cells. transfer DNA (T-DNA) insertional mutations in the PDI9 gene disrupted pollen viability and development in plants exposed to heat stress. In particular, the pollen grains of pdi9 mutants exhibited disruptions in the reticulated pattern of the exine and an increased adhesion of pollen grains. Pollen in the pdi10 single mutant did not display similar heat-associated defects, but pdi9 pdi10 double mutants (DMs) completely lost exine reticulation. Interestingly, overexpression of PDI9 partially led to heat-associated defects in the exine. We conclude that PDI9 plays an important role in pollen thermotolerance and exine biogenesis. Its role fits the mechanistic theory of proteostasis in which an ideal balance of PDI isoforms is required in the endoplasmic reticulum (ER) for normal exine formation in plants subjected to heat stress.
Highlights
Pollen has the most structurally complex cell wall produced by plants and serves as a highly protective barrier for the male gametophyte
Phenotypic characterization of mutants harboring transfer DNA (T-DNA) insertions in the two protein disulfide isomerases (PDIs)-M genes of Arabidopsis, PDI9 and PDI10, revealed that the pollen of pdi9 loss-of-function mutants were hypersensitive to heat stress, as indicated by a pronounced decrease in pollen viability in comparison to WT control plants grown under identical conditions
GUS reporter experiments indicated that the promoters of both PDI9 and PDI10 were active in anthers and pollen, the pollen of pdi10-1 single mutants did not exhibit a similar susceptibility to heat stress as the pdi9-1 and pdi9-2 mutants
Summary
Pollen has the most structurally complex cell wall produced by plants and serves as a highly protective barrier for the male gametophyte. The composition of the pollen cell wall changes throughout development and is precisely regulated by a transcription factor cascade (Xu et al, 2014; Lei et al, 2017), and the coordinated synthesis of biosynthetic enzymes secreted from the endoplasmic reticulum (ER; Lallemand et al, 2013). Developing pollen is surrounded by a layer of highly metabolically active sporophytic anther cells designated the tapetum, which play a critical role in pollen wall biogenesis by synthesizing and secreting the sporopollenin monomers used to form the exine layer (Ahlers et al, 1999; Grienenberger et al, 2010; Quilichini et al, 2015; Battat et al, 2019). The programmed cell death of the tapetum detaches it from the pollen cell wall, simultaneously depositing the final layer of sporopollenin (and in some species, lipid), causing the dehiscence of mature pollen grains from the anthers (Kawanabe et al, 2006; Parish and Li, 2010)
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